The sequence arrangement of an eucaryotic organelle DNA will be determined by mapping with restriction endonuclease. Fragments produced by maximum cleavage with EcoR1* restriction endonuclease will be identified. Partial enzymatic hydrolysis with EcoR1 will be employed to prepare fractions that contain more than one restriction fragment. Each partially digested fraction will be maximumly cleaved with EcoR1 and analyzed to identify the restriction fragments in each fraction. Comparison of the restriction fragments in each fraction will allow assignment of the fragment sequence in the circular DNA molecule. An alternative method for determining the sequence of restriction fragments may be preparation of fragments with two different restriction endonucleases and ascertaining the overlapping fragments. Restriction fragments will be isolated by preparative electrophoresis in agarose gels. Location of genes on the restriction map will be initiated by identifying fragments that code for rRNA and locating the site for initiating DNA replication. Hybridization of rRNA to DNA on membrane filters will be employed to identify rRNA genes. High specific activity rRNA will be prepared by iodination with I125; therefore, hybridization assays will require less than micro gram amounts of each restriction fragment. The origin of DNA replication on the restriction fragment map will be determined by electron microscopic identification of fragments that contain replication loops.